Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with short life expectancy and no effective therapy. We previously identified upregulated miR-124 in NSC-34-motor neurons (MNs) expressing human SOD1-G93A (mSOD1) and established its implication in mSOD1 MN degeneration and glial cell activation. When anti-miR-124-treated mSOD1 MN (preconditioned) secretome was incubated in spinal cord organotypic cultures from symptomatic mSOD1 mice, the dysregulated homeostatic balance was circumvented. To decipher the therapeutic potential of such preconditioned secretome, we intrathecally injected it in mSOD1 mice at the early stage of the disease (12-week-old). Preconditioned secretome prevented motor impairment and was effective in counteracting muscle atrophy, glial reactivity/dysfunction, and the neurodegeneration of the symptomatic mSOD1 mice. Deficits in corticospinal function and gait abnormalities were precluded, and the loss of gastrocnemius muscle fiber area was avoided. At the molecular level, the preconditioned secretome enhanced NeuN mRNA/protein expression levels and the PSD-95/TREM2/IL-10/arginase 1/MBP/PLP genes, thus avoiding the neuronal/glial cell dysregulation that characterizes ALS mice. It also prevented upregulated GFAP/Cx43/S100B/vimentin and inflammatory-associated miRNAs, specifically miR-146a/miR-155/miR-21, which are displayed by symptomatic animals. Collectively, our study highlights the intrathecal administration of the secretome from anti-miR-124-treated mSOD1 MNs as a therapeutic strategy for halting/delaying disease progression in an ALS mouse model. Amyotrophic lateral sclerosis (ALS) is a fast progressive disease that results from the degeneration of the upper and lower motor neurons (MNs) in the motor cortex, brainstem, and spinal cord (SC). Consequently, this neurodegeneration leads to voluntary muscle weakness and wasting, resulting in a diversity of symptoms, such as dysarthria, dysphagia, weakness, and atrophy of the limbs. The pathological mechanisms underlying the disease include misfolded protein aggregation, mitochondrial dysfunction, dysregulated axonal transport, altered synaptic dynamics, excitotoxicity, and neuroinflammation . It is nmicroRNAs (miRNAs) have been defined as important regulatory molecules for gene expression, and their dysregulation in ALS can be modulated toward the recovery of cell function and an increase in mSOD1 mouse lifespan, as reviewed in . FurtherFor this reason, we decided to evaluate the therapeutic potential of anti-miR-124-treated mSOD1 MNs in preventing disabilities in mSOD1 mice. Thus, we performed an intrathecal injection of the preconditioned secretome in the mSOD1 mice at the early symptomatic disease stage. At the symptomatic stage, we evaluated the motor performance by testing the gait quality and the corticospinal function. Moreover, we collected the gastrocnemius muscle for muscle integrity evaluation and lumbar SC for neurodegeneration, neuroinflammation, and myelination assessments. Our results revealed that the preconditioned secretome prevented MN and glial pathological mechanisms and improved motor disabilities in the ALS mice. Therefore, this preconditioned secretome shows promise as a therapeutic tool to be tested in stratified patients with upregulated miR-124 levels, namely at symptom onset, contributing to the advance of precision medicine.l-glutamine, no sodium pyruvate, supplemented with 10% FBS ; 1% penicillin/streptomycin or pen/strep ; and 0.1% geneticin sulphate 418 (G418) for cell selection), as described in [® Anti-miRTM miRNA inhibitor, #AM10691) and mixed with X-tremeGENE™ HP DNA Transfection Reagent  in a proportion of 2:1 and diluted in Opti-MEM™. Cells were left for 12 h, then fresh differentiation medium was added, and cells were maintained for an additional 48 h (4 DIV). This medium containing the factors secreted by mSOD1 MNs (secretome) was collected and centrifuged at 1000× g for 10 min to remove any cell debris. Since intrathecal injection requires very low volumes of the secretome [g at 4 °C for 180 min and then stored at −80 °C until being used in the intrathecal injection. In parallel and to confirm the successful entry of the injected secretome into the lumbar SC, we labelled the sEVs derived from the secretome of 4 DIV WT NSC-34 MNs and injected them intrathecally in the WT mice  at 100,000× g for 2 h. Finally, we labelled the sEVs with a PKH67 fluorescent linker kit  in accordance with the manufacturer’s specifications and resuspended them in DMEM-F12 plus 1% FBS depleted from the sEVs.NSC-34 MN cell line stably transfected with human WT SOD1 and human mSOD1 was grown in proliferation media  at 22–24 °C, and 55% humidity) and provided with food and water ad libitum. The colony was maintained on a background B6SJL by breeding mSOD1 transgenic males with non-transgenic females. Transgenic mSOD1 mice were compared to aged-matched WT mice. The procedures performed were in accordance with the European Community guidelines  and the Portuguese Laws on Animal Care . All the protocols used in this study were approved by the Portuguese National Authority  and Ethical Subcommittee in Life and Health Sciences .Mice were purchased from The Jackson Laboratory , namely transgenic B6SJL-TgN mSOD1Gur/J males (no. 002726) and non-transgenic B6SJLF1/J wild-type (WT) females. They were housed at the animal facility of the Life and Health Science Research Institute (ICVS)The sequence of the experiments developed in this study is graphically summarized in ®, Pfizer, Inc., Brooklyn, NY, USA) for the reversal of the anesthesia [Experiments to assess the therapeutic efficacy of the anti-miR-124-treated mSOD1 MNs were performed in the mSOD1 mice injected with the concentrated preconditioned secretome. The 12-week-old animals (early symptomatic stage of the disease) were anesthetized intraperitoneally (i.p.) with ketamine (75 mg/kg) plus medetomidine (0.5 mg/kg) . Once anesthesia . The WT esthesia  and the esthesia . Additioesthesia ,30. ThenNote that no statistical differences were found in the mouse weight (g) between WT and transgenic animals, before or after treatment .To determine the impact of the secretome from anti-miR-124-treated mSOD1 MNs on motor behaviour, we evaluated motor performance in the animals two weeks after the injection. We assessed the gait quality by the footprint test, the muscular strength by the hanging wire test, and the spontaneous activity by the cylinder test. Finally, the deficits in the corticospinal function were also examined using the limb clasping and grasping tests.The fore and hind paws of mice were painted with non-toxic dyes of different colours, and the mice were placed on absorbent paper in an inclined corridor so that they would walk up it since mice have the tendency to run upwards to escape . We measThe mice had to remain clinging to an inverted surface of a cage lid, demonstrating their grip strength . The durThe mice were placed into a clear cylinder, and the number of times they reared up against the cylinder wall  was counThe mice were suspended by holding their tails, and the position of their hindlimbs and toes was observed for several seconds. An abnormal phenotype is represented by the retraction of the hindlimbs toward the abdomen (clasping reflex)  and/or tTo avoid the animal suffering associated with the disease phenotype, we sacrificed the animals at 15 weeks of age (one week after the beginning of the behaviour tests). Mice were anesthetized i.p. with ketamine (75 mg/kg) plus medetomidine (0.5 mg/kg) and transcardially perfused with 0.1 M PBS at pH 7.4. Then, lumbar SC and left gastrocnemius muscle were dissected and rapidly frozen at −80 °C for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) assays. For histological and immunohistochemistry studies, animals were perfused with a fixative containing 4% paraformaldehyde in PBS, and the lumbar SC and left gastrocnemius muscle were removed. Then, the lumbar SC was post-fixed with the same fixative for 24 h at 4 °C and preserved in 30% sucrose solution. The gastrocnemius muscle was stretched over a notched wooden stick to maintain the in vivo length and to prevent over-contraction. Later, the muscle was frozen in isopentane cooled over liquid nitrogen and stored at −80 °C .® O.C.T. Compound  before transversal 20 μm thick sections were serially cut using the cryostat Leica CM1900 . The sections were collected on Superfrost Plus glass slides  and preserved at −80 °C.For sectioning, the muscle was sliced directly in the cryostat, while the SCs were first embedded in Tissue-TekThe lumbar SC and muscle were homogenized in TRIzol reagent  using a Pellet Mixer . The muscle homogenization in TRIzol reagent was performed by cutting the tissue into small pieces, followed by transferring the resulting mixture through a 1 mL syringe with a 20 G needle until a homogeneous mixture was obtained. The total RNA was extracted and quantified on a NanoDrop ND100 Spectrophotometer  according to the standard procedure in our laboratory .For gene expression, the total RNA was reverse transcribed into cDNA using the Xpert cDNA Synthesis Supermix kit . RT-qPCR was performed using an Xpert Fast SYBR Mastermix BLUE kit (GRiSP) and the primer sequences listed in TM RT Kit  and RT-qPCR using PowerUp™ SYBR™ Green Master Mix  and the primer sequences listed in For miRNA expression, cDNA was performed using a miRCURY LNA−ΔΔCt method relative to that of the endogenous control. Each sample was measured in duplicate. The cDNA synthesis was performed in a thermocycler , and the RT-qPCR was run on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems).The expression of mRNA/miRNA was measured via the 2The gastrocnemius muscle was stained with hematoxylin and eosin using an AutoStainer XL (Leica). Briefly, the muscle sections were rinsed in distilled water and placed into Harris hematoxylin solution for 1 min, followed by a tap-water wash for 2 min. Then, differentiation with 0.5% ammonia water was performed for 10 s. After washing with tap water for 2 min, the sections were stained with eosin for 20 s; dehydrated using 70, 95, and absolute alcohol for 2 min each; and covered with xylol for 1 min. The samples were mounted with a glass coverslip using Entellan mounting media . Photos were obtained using an Olympus widefield inverted microscope IX53 with 20× magnification. The images from three fields of the same muscle section per animal  were analysed using Fiji software. We measured the mean area occupied by each fiber from the transversal section of the muscle.To evaluate neurodegeneration, the lumbar SC was stained with Fluoro-Jade B . The samples were defrosted at RT for 10 min. Then, they were incubated in 0.06% potassium permanganate solution for 30 min with gentle agitation. After a 5 min PBS rinse, the samples were stained with a 0.001% solution of Fluoro-Jade B dissolved in 0.1% acetic acid vehicle for 30 min with mild agitation. The samples were then rinsed through three changes of PBS for 3 min each with agitation and mounted in Fluromount-G (Sigma-Aldrich) with a glass coverslip on the top.To access the distribution of sEVs in the SC, we also stained the neurons and astrocytes with NeuN and glial fibrillary acidic protein (GFAP), respectively. The presence of glial cells in the lumbar SC was also evaluated through the quantification of ionized calcium-binding adaptor molecule 1 (Iba-1) for microglia and GFAP for astrocytes. The previously described protocol was followed with minor modifications . After dThe fluorescence images were obtained in a Leica DMi8-CS inverted microscope with Leica LAS X software, and the different z-stacks were merged and analysed with Fiji software. We measured the mean fluorescence of the ventral horn of one lumbar SC transversal section per animal (N = 3 per group) stained with Fluoro-Jade B and obtained with 20× magnification. Images stained with Iba-1 and GFAP were obtained with 40× magnification and analysed from five ventral horn fields per animal . We measured the fraction of the area occupied by the GFAP- and Iba-1-positive cells.TM FL1500 Imaging System. Bands were quantified using iBrightTM analysis software. Results were normalized to the expression of β-actin and indicated as fold change.Total protein was isolated from the organic phase of the TRIzol–chloroform from lumbar SC and muscle  and quanp > 0.05), one-way ANOVA was applied, followed by multiple-comparisons Bonferroni post hoc correction, as well as unpaired and parametric Student’s t-tests. Welch’s t-test correction was applied when variances were different between the two groups. Values of p < 0.05 were considered statistically significant. Data were expressed as mean ± SEM values, except for the non-continuous categorical variables. Results were analysed using GraphPad Prism 8.0.1 .Non-parametric ANOVA  and unpaired and non-parametric Mann–Whitney tests were used when the data showed non-normal distribution. Non-continuous categorical variables were analysed using the chi-square (and Fisher’s exact) test. For data with normal distribution  after the intrathecal injection for both periods of time A,B. To fFrom here on we decided to use the whole secretome, and not only the injection of sEVs, considering its advantages for ALS therapeutics. On the one hand, storing and administering the secretome is easier than doing so for sEVs and facilitates their preservation. On the other hand, the secretome also contains additional bioactive molecules that may increase its neuroprotective potential, as a consequence of the effects of anti-miR-124 on MN survival . When coIn our prior studies, we demonstrated that miR-124 levels in the secretome recapitulate the levels observed in cells, either non-modulated or treated with its mimics and inhibitors ,10,11,13p < 0.01). The injection of the secretome from anti-miR-124-treated MNs, under the same conditions, was successful in downregulating the expression of miR-124 . These observations are associated with an abnormal gait quality, muscle weakness, and less spontaneous vertical activity, respectively. In addition, a large percentage of mSOD1 animals showed enhanced limb clasping (p < 0.01 vs. WT + vehicle) and grasping (p < 0.0001 vs. WT + vehicle) reflexes, in accordance with abnormalities in the corticospinal tract.As indicated in p < 0.0001 vs. mSOD1 + vehicle) and grasping reflexes (p < 0.01 vs. mSOD1 + vehicle) was similarly clear 2 weeks after the treatment with the secretome from anti-miR-124-treated mSOD1 MNs.The secretome from anti-miR-124-treated mSOD1 MNs was able to inhibit such impairments. We noticed improvements in all the assessed behavioural tests E–G, thoup < 0.0001 vs. WT + vehicle, p < 0.05 vs. WT + vehicle, Since we observed motor performance disabilities in the mSOD1 mice, a common symptom of ALS caused by muscle weakness , alreadyp < 0.01 vs. mSOD1 + vehicle, p < 0.05 vs. mSOD1 + vehicle, Notably, 3 weeks after the intrathecal injection of the preconditioning secretome from anti-miR-124 mSOD1 MNs in the ALS mice, we noticed the reversal of the skeletal muscle atrophy  when assessed in the lumbar SC of the mSOD1 mice at 15-week-old. These results revealed an impairment of the postsynaptic dynamics, retrograde/anterograde axonal transport, and CX3CL1 that may have compromised paracrine signalling with the microglia receptor CX3CR1. Neurodegeneration was further confirmed by the decrease in the protein NeuN .Neuronal survival and axonal preservation depend on myelin integrity , an issup < 0.05 vs. mSOD1 + vehicle, p < 0.05 vs. mSOD1 + vehicle, The intrathecal injection of the preconditioned secretome exerted protective effects against most of the observed impairments. In fact, we observed an upregulation of PSD-95 and NeuN (including the protein expression) , known to exacerbate neuropathological and behavioural deficits [p < 0.01 vs. WT + vehicle, p < 0.001 vs. WT + vehicle) and the milk fat globule epidermal growth factor 8 (MFG-E8) (p < 0.05 vs. WT + vehicle), which have been shown to compromise phagocytic ability [p < 0.05 vs. WT + vehicle), which have been shown to regulate neuroinflammation [p < 0.05 vs. mSOD1 + vehicle, p < 0.05 vs. mSOD1 + vehicle), which are associated with a reparative phenotype.Concerning the categorization of microglia immunoreactivity, we found the downregulation of arginase 1 in the mSOD1 SC 3 weeks after vehicle injection in the early symptomatic mice . Note the non-existence of alterations in the gene expression of S100 calcium-binding protein B (S100B) in the absence or presence of the preconditioned secretome.Interestingly, the preconditioned secretome led to a significant reduction in these parameters , which may have compensated for each other to reach a steady state. Overall, our results highlighted that the preconditioned secretome regulated the lumbar SC immunoreactivity.When we assessed the dysregulation of genes associated with inflammation, we observed an imbalance with decreased iNOS and interleukin (IL)-10, together with elevated levels of tumour necrosis α/TNF-α , miR-155 (p < 0.01), and miR-124 (p < 0.05), when compared with the mSOD1 mice injected with the vehicle  , and comCell secretomes are rich in soluble proteins and sEVs , which rIn previous studies, we found upregulated miR-124 in the SC of mSOD1 mice , which wBehavioural tests were performed on 14-week-old animals, and molecular assessments of the SC were carried out after 15-week-old mouse sacrifice, which preceded the advanced symptomatic stage that occurs at 16 weeks of age . No apprThe motor symptoms observed in ALS mice were unequivocally associated with muscular atrophy, the denervation of the neuromuscular junction, and the degeneration of the MNs. We observed a reduction in the area of gastrocnemius muscle fibers, together with MN loss and a lack of synaptic activity. Such a loss of muscle integrity is usually a result of MN demise ,87. HoweWe observed high levels of neurodegeneration and MN loss in the lumbar SC of symptomatic ALS mice, as previously published . To contDysfunctional oligodendrocytes and myelin structures have been reported in the SC of ALS mouse models and patients, as reviewed in . In accoMicroglia activation and astrocyte reactivity contribute to the dissemination of neurodegeneration ,50. HoweWe observed in a previous study that symptomatic mSOD1 mice also exhibited upregulated levels of miR-155 and miR-146a, alongside miR-124, in the SC, which are known to contribute to neuroinflammation . FurtherOverall, this study validated the therapeutic potential of the secretome derived from anti-miR-124-treated mSOD1 MNs in the prevention of pathological neural cell signatures in symptomatic mSOD1 mice and the subsequent dysregulation of the paracrine signalling implicated in disease dissemination. From a single intrathecal injection of the preconditioned secretome carrying basal levels of miR-124 at the early symptomatic stage of the mSOD1 mice, we were able to regulate miR-124 toward normal values. With such an approach, we sustained anti-inflammatory/phagocytic microglia, MN function, myelination, astrocyte neuroprotection, and immune regulation at 3 weeks after injection, i.e., at the symptomatic stage wherein all these mechanisms turn pathological. Importantly, the secretome also preserved synaptic signalling, functional skeletal muscle, and neuromuscular junction, which translated to the preservation of motor coordination, balance, corticospinal function, and spontaneous behaviour in the symptomatic mSOD1 mice. Our preconditioned secretome based on the strict regulation of miR-124 levels showed immunoregulatory and potential neuroregenerative properties, opening a new avenue for developing a novel effective therapeutic strategy that can be translated into clinical applications in the future. To achieve this, it will be crucial to stratify patients with upregulated levels of miR-124 based on MNs differentiated from induced pluripotent stem cells (iPSCs) or neural precursor cells generated from their somatic cells. In this sense, we propose the modulation of upregulated miR-124 expression levels in such MNs to produce the preconditioned personalized secretome. We can envisage, as was lately described for the secretome of iPSCs , that ou


In vitro drug release was performed by UV spectrophotometry, dialysis, and vial bag methods. Cytotoxicity assessment of HA and zinc-oxide gels was performed in human periodontal ligament fibroblasts (HPdLF) and human gingival fibroblasts (hGFs). An inverted phase-contrast microscope was used to assess the morphological changes. At 24 and 48 hr, HPdLF cells showed the highest viability in 0.1% low molecular weight-HA (LMW-HA) with a median value of 131.9, and hGFs showed the highest viability in 5% LMW-HA with a median of 129.56. The highest viability of HPdLF cells was observed in 5% high molecular weight-HA (HMW-HA), with a median value of 127.11. hGFs showed the highest viability in 1% HMW-HA with a median value of 97.99. Within the limitations of the present study, we concluded that LMW-HA is more efficient than HMW-HA. Both HPdLF and hGF cells showed complete cell morbidity with zinc-oxide hydrogels. Therefore, zinc oxide-based gels in concentrations as low as 9% could be toxic intraorally to soft tissues that harbor gingival and periodontal ligament fibroblasts.Hyaluronic acid (HA) is a naturally occurring biopolymer, with a remarkable wound healing property. Zinc-oxide non-eugenol is a material widely used for periodontal dressing in dentistry. However, it has been reported that zinc oxide non-eugenol is toxic to osteoblasts and fibroblasts. Hence, the present study aimed to evaluate the drug release and cytotoxicity of HA and zinc-oxide gels. Hydrogels of HA and zinc oxide were formulated with carbopol as a carrier.  Bacterial infections, functional and physical forces in the oral cavity during periodontal surgery may affect healing . Thus, iHyaluronic acid (HA) is an innately occurring high molecular weight polysaccharide formed in the human body as a part of a natural wound healing mechanism. HA acts as an essential element of cell migration and proliferation, thus regulating tissue hydrodynamics . In addiZinc oxide non-eugenol is a widely used material for periodontal wound dressings in dentistry. The antibacterial reaction from metal oxide and fatty acids acts as a barrier to protect wounds ,16. Topiin vitro studies have reported the high toxicity of zinc-oxide non-eugenol to osteoblasts and fibroblasts [Despite the usage, several roblasts . Studiesroblasts ,21. FurtHA is available in two forms ; a low min vitro drug release of HMW-HA and LMW -HA and compare their cytotoxicity with zinc oxide gels. The cytotoxicity assessment was performed using human periodontal ligament and gingival fibroblasts.Unfortunately, the number of studies on wound healing benefits of HA are very scarce, and it is mostly used in either a gel or a liquid form. The most common concentrations used are 0.2, 0.8, and 1%, and they were also commercially formulated from HMW-HA. Studies that used LMW-HA variants are also limited. Gingigel, Hyadent, Hyalugel, Aminogam, and Klirich are a few commercially available products that are sparsely available and are of different concentrations formulated with HMW-HA. As a clinician, choosing these commercially available forms of HMW and LMW-HA for periodontal therapy needs more evidence. Therefore, the present study aimed to evaluate the in vitro studies [The present study is a two-way factorial design. The study protocol is as per CRIS guidelines for  studies . The stuGel base was prepared using carbopol 940 (manufactured by Sigma-Aldrich) by soaking in water overnight. 0.1% w/v concentration of carbopol gel was prepared with suitable dilution in water.HA gel was prepared by dissolving an appropriate amount of HA in 0.1% w/v carbopol gel. The pH of the gel was adjusted to 7.0 using triethanolamine.Zinc oxide gel was prepared by dissolving an appropriate amount of zinc oxide in 0.1% w/v carbopol gel.Gel was placed in a vial, and pH 7.4 phosphate buffer saline was added. The mixture was kept in a shaking incubator at 37 ± 2°C with 100 rpm. At a predetermined time interval, the sample was collected and centrifuged. We observed that HMW-HA settled down along with the Carbopol due to its high molecular weight, and no HA was present in the supernatant. So, we were not able to perform the release study for the prepared gels using HMW-HA.The molecular weight of HA used was 8 kDa. The cut-off of the dialysis bag was 12 kDa. The sample was collected and centrifuged in a shaking incubator at 37 ± 2°C with 100 rpm at a predetermined time interval.Different concentrations of LMW-HA were prepared in water and analyzed using a UV spectrophotometer at a wavelength of 205 nm. The calibration curve was plotted as a function of absorbance Vs. LMW-HA concentration. The samples of HA and zinc oxide hydrogels were sterilized by gamma irradiation  at a dose rate of 2.68 kGy/hr.2 incubator. The dead, floating cells were removed by changing the media every two days. Then, the hGFs and HPdLF cells were exposed to the test and control agents. The morphological changes were observed using an inverted phase-contrast microscope and compared at 24, 48, and 72 hr. MTT assay was performed to evaluate the cytotoxicity, and it was matched between the following groups.The human gingival fibroblasts (hGFs) and periodontal ligament cells (HPdLF) were cultured after procuring tissue fragments from the extracted tooth by the explant technique as described earlier in a study by Kwon et al. . The culSPSS-22 software was used for statistical analysis. The Kruskal–Wallis test was used to analyze the effective concentration in the HA group. In addition, a comparison between all the different concentrations of HA for the viability of cells was performed for both hGFs and HPdLF at three different time points, i.e., 24 hr, 48 hr, and 72 hr by using the Friedman test.in vitro release study results showed that 0.1% LMW-HA gel was released only at 1, 2, 3, and 4 hr, and there was no release of HA after 4 hr for 0.1% LMW-HA.The drug release study was performed for LMW-HA using the dialysis bag method. The amount of drug released for 0.1% LMW-HA was only 4 hr. After 4 hr timeline, no release was noted. All the other three concentrations of 1%, 5%, and 10% showed drug release for 72 hr, and an increasing trend with time was noticed. The evaluation of The other three concentrations released HA gel for up to 72 hr. There was an increase in drug release as the HA concentration increased from 1% to 10%. The release was recorded for up to 72 hr. Drug release patterns are illustrated in All the four concentrations of zinc oxide gel used, i.e., 87%, 36%, 18%, and 9% (w/v), showed total cell mortality in both hGFs and HPdLF cells.At 24 and 48 hr, the LMW-HA group for HPdLF cells showed the highest viability at 0.1% with a median value of 131.9, and for hGFs, it was 5% with a median of 129.56. For the HMW-HA group, the highest viability for HPdLF cells with a median value of 127.11 was noted in 5% HMW-HA, and for hGFs cells, it was pointed out in 1%, as they showed the highest viability with a median value of 97.99. The HMW-HA group of HPdLF cells and hGFs showed statistically significant results (p < 0005). At 72 hr, it was observed that LMW-HA for both HPdLF and hGFs, and HMW-HA HPdLF cells showed statistically significant results (p < 0.005). A detailed cell viability analysis of HMW-HA and LMW-HA groups at 24, 48 and 72 hours is given in The analysis of the viability of HPdLF cells and hGFs at 24 hr indicated that the highest percentage of viable HPdLF cells were observed in 0.1% LMW-HA and 5% HMW-HA. In the case of hGFs, the highest percentage of viable cells were found in 5% LMW-HA and 1% HMW-HA. The lowest percentage of viable cells in all the groups was 10%, suggesting it was ineffective for all the cells in both LMW and HMW variants . The HMWConversely, at 72 hr, it was noticed that HMW-HA had slightly varied results compared to the other two timelines. The highest percentage of viable HPdLF and hGFs were found in 0.1% HMW-HA. LMW-HA showed similar results at 24- and 48-hr timelines, i.e., for HPdLF cells, 0.1% was found to be effective and 5% for hGFs cells. The results of HPdLF and hGFs cells of LMW-HA and HPdLF cells of HMW-HA groups showed statistically significant results (p < 0.005). The comparison of viability values within the groups at subsequent time intervals by the Friedman test showed statistically significant changes for HPdLF cells in 0.1% HMW-HA, which showed the highest viability percentage at all the timelines. 10% HMW-HA and LMW-HA showed the statistically significant (p < 0.005) least viability percentage for all the groups except HPdLF cells in 0.1% LMW-HA. Table 3.CA comparison of viability among the groups using the Kruskal–Wallis test indicated that 10% of LMW-HA and HMW-HA had shown  almost negligible percentage of viable HPdLF cells and hGFs at all the three timelines of 24, 48, and 72 hr .During the cytotoxicity assay, any changes in the morphology of HPdLF cells and hGFs were observed using an inverted phase-contrast microscope. The images of 96-well microplates used for the assay are presented in in vitro are limited. The present study is one of the few to evaluate the in vitro release of LMW-HA. Quantification of HMW-HA was difficult due to its high molecular weight and complex molecular structure [The investigations that focused on drug release patterns of HMW and LMW-HA tructure . In the In Group A, 87%, 36%, 18%, and 9% (w/v) of the samples used were zinc oxide-based hydrogels. We had chosen 87% and 36% (w/v) concentrations from the existing evidence of commercially available zinc oxide-based periodontal packs . The oth26At the 24-hr timeline, for HPdLF cells, LMW-HA showed viabilities in the following order: 0.1%>1%, then, 5%>10%, suggesting 0.1% to be more effective and 10% to be least effective. This finding indicates that 0.1% has a physical property, suitable for HPdLF cells, which might have favored the proliferation of cells. On the other hand, HMW-HA gels showed viabilities differently, i.e., 5%>1%>0.1%>10%. These trends in viability indicate that the most effective concentration is 5% HMW-HA for HPdLF cells. This could be because of the mucoadhesive property of high molecular weight, which can stay longer with the HPdLF cells.hGFs exhibited the viability to LMW-HA in the order of 5%>1%>0.1%>10% and to HMW-HA in the order of 1%>5%>0.1%>10%. Hence, the effective concentrations of LMW-HA and HMW-HA for hGFs were 5% LMW-HA and 1% HMW-HA, respectively. It can be inferred that the liquid-type consistency of 5% LMW-HA and 1% HMW-HA is favourable for the survival and proliferation of hGFs, unlike the HPdLF cells. In all the groups, 10% concentration was observed to be ineffective with the lowest viability percentage. This ineffectiveness of 10% HA gel can be attributed to the rheological properties of HA. This was in correlation with previous studies on the rheological properties of HA ,31–36. TThe effectiveness of LMW-HA over HMW-HA could be related to its immunoregulatory property, which is usually noticed at the sites of inflammation. LMW-HA is formed from enzymatic degradation of HMW-HA. LMW-HA promotes the production of immune mediators thereby stimulating an immune response. This could enhance wound healing better than the high molecular weight variant . Hence, in vitro on human skin cell lines [A similar pattern of gamma irradiation was performed to prepare LMW-HA and found that it showed statistically significant benefits of wound healing ll lines .In another randomized placebo-controlled clinical trial, there were no statistically significant benefits of HA-applied sites after surgery compared to placebo sites . This maThe effective concentration of HMW-HA for hGFs is found to be 1%. Above 1% concentration is inhibitory to hGF cells.The effective concentration of HMW-HA for HPdLF cells is found to be 5%.The effective concentration of LMW-HA for hGFs is found to be 5%.The effective concentration of LMW-HA for HPdLF cells is found to be 0.1%LMW-HA is found to be more effective than HMW-HA in both HPdLF cells and hGFs.The mortality of both HPdLF cells and hGFs in vitro to concentrations even as low as 9% of zinc oxide- based gels have shown to be very toxic. Hence, exposing this material intraorally to soft tissues that harbor gingival and periodontal ligament fibroblasts is questionable.After a comprehensive analysis of the results, we could arrive at a conclusion that


E)-8-dodecenyl acetate, which may possibly be a component in the pheromone blend for GEM. Exposure to these compounds in the laboratory reduced flight capacity. Our results suggest these two compounds could be included in monitoring or management programs for the Giant Eucosma moth.Rosinweed is a novel perennial crop being developed for oilseed and biofuel in the midwestern US. A primary pest is the Giant Eucosma Moth (GEM). Little is known about the chemical ecology or flight behavior of adults, but many attractants have been identified from other closely related species. The goals of this study were to evaluate whether any of these compounds could improve the capture of GEMs on sticky cards in the field and determine whether the most attractive volatiles might affect flight behavior in the laboratory. We found that there was significant attraction to . Little is known about the chemical ecology or flight behavior of E. giganteana, but many semiochemicals have been identified from other closely related Eucosma species. Some of these compounds include: (Z)- and (E)-8-dodecenyl acetate, (E)-9-dodecenyl acetate, (Z)-8-dodecenol, -8,10-dodecadienyl acetate, and -9,12-tetradecadienyl acetate. The goals of this study were to evaluate whether any of these compounds could improve capture of E. giganteana on clear sticky cards in the field, and the most attractive volatiles might affect flight behavior on a computer-automated flight mill assay. We found that there was significant attraction to (E)-8-dodecenyl acetate in two years in the field, which may possibly be a component in the pheromone blend for E. giganteana. On flight mills, E. giganteana flew an average of 23 km in a 24 h period. The presence of attractive stimuli -8-dodecenyl acetate) had arresting properties and decreasing flight distance on the mill by 78 to 80%. The longest flight distances were registered in the morning (4:00–12:00) and were 1.8-fold greater than flight distances and durations at night (20:00–4:00). (E)-8-dodecenyl acetate may be useful in behaviorally based monitoring and management strategies for E. giganteana. Overall, our research expands the knowledge on the chemical ecology of adult E. giganteana. Silphium integrifolium Michx., or silflower, is a perennial plant that is native to the prairies of North America [S. integrifolium and enhance seed yield, so it can substitute for current annual oilseed crops .Eucosma giganteana (Riley) (Lepidoptera: Tortricidae), a specialist of Silphium spp. This species is thought to be univoltine, with adult flight taking place during early to mid-summer in Kansas, coinciding with the time that silflower blooms . Adults mate and lay eggs on inflorescences, then the larvae hatch and bore into the heads of the flowers. After feeding on the flower heads for several weeks, the larvae then descend and burrow into the root crowns, where they feed and weaken the plant’s ability to store carbohydrate reserves for future years [E. giganteana reduced seed production in infested flower heads by 45–85% versus undamaged heads [S. integrifolium in and around Kansas [E. giganteana in North Dakota eliminated viable seeds in nursery plants of the closely related S. perfoliatum [S. integrifolium, effective pest management tactics are imperative.One of the primary pests in this system is re years . Prior wd Kansas . Furtherfoliatum . Given tE. giganteana. Vilela et al. [E. giganteana colonization by trimming whole S. integrifolium plantings to shift their flowering date to later in the season; while the flowering date was shifted, this did not result in decreased pest infestations. A single application of 0.4% permethrin provided a three-fold reduction in E. giganteana infestation compared to untreated controls. Nonetheless, permethrin has broad spectrum activity against many taxa, including pollinators, and many pollinators use S. integrifolium as a resource [S. integrifolium has some ability to self-pollinate, its seed yield benefits greatly from cross-pollination [A variety of approaches have been evaluated for the management of a et al.  unsuccesresource . Additiolination . As a reE. giganteana. For example, the sex pheromone is unknown for this species, and no one has evaluated attractants, repellents, or other semiochemicals for this pest, either in the field or laboratory. However, a great deal more is known about related Eucosma species. For example, the pheromone of Eucosma nothanthes was isolated and discovered to be (Z)-8-dodecenyl acetate and (Z)-8-dodecenol [E,E)-, -, -, and -8,10-dodecadien-l-yl acetate were classified as conserved sex pheromone components or attractants, including for Eucosma spp. [E)-9-dodecenyl acetate, (Z)-9-dodecenyl acetate, and -8,10-dodecadienyl acetate captured Eucosma bobana Kearfott, Eucosma ponderosa Powell, and Eucosma recissoriana complex [Eucosma cana Haw. and Eucosma cumulana Guin. were attracted to and trapped by lures with (E)-8-dodecenyl acetate, (Z)-8-dodecenyl acetate, and (E)-8-dodecen-1-ol in apple orchards. The structural commonality in attractants for related species suggests that they may also exhibit behaviorally relevant activity for E. giganteana.One class of alternative IPM tactics are novel semiochemical-based monitoring and behaviorally based management approaches, whereby pest populations are manipulated at a distance with semiochemicals and removed from the foraging population with targeted insecticide use or by virtue of their biology . Overallodecenol . Furthersma spp. . A combi complex . Further complex  found thE. giganteana, including its baseline flight capacity. In other Lepidopterans, flight capacity may be surprisingly high for small-sized species in the same family. For example, female flight capacity for female Cydia pomonella L. (Lepidoptera: Tortricidae) can reach up to 11 km [Ephestia kuehniella Zeller  [Cactoblastis cactorum (Berg) , during dusk and the beginning of the scotophase [E. giganteana, adult diurnal activity patterns, and its flight responses to semiochemicals all remain open questions.Finally, there is very little known about the flight dynamics of to 11 km . Importaralidae) . Flight otophase . Thus, tE. giganteana to semiochemicals identified from congeneric Eucosma when paired with clear sticky traps, and determine how the most attractive ones affect flight capacity in the laboratory. These compounds may be useful in behaviorally based monitoring and management strategies. We hypothesized that attractive semiochemicals in the field would reduce flight capacity in the laboratory. Overall, we expect that the knowledge gained from this study would greatly expand our understanding of the behavioral and chemical ecology of E. giganteana.The goal in the current study was to evaluate attraction of S. integrifolium breeding plots in East-Central Kansas at The Land Institute , perennial sorghum, and blends of fescue.All traps were deployed at three fields of actively managed nstitute . Fields E. giganteana adults were used for laboratory data collection because it is not currently possible to rear the species in the laboratory. Individuals were collected from a combination of two UV light traps deployed at The Land Institute in Salina, KS, USA, and hand-collected at night during the period of peak activity for E. giganteana. Individuals were collected on host silflower plants from 20:30–22:00 from multiple field sites between June and August in 2019 and 2020. Both sexes were equally represented from collections on flowers. Adults were held no more than 24 h at constant temperature (23 ± 0.1 °C) and 60 ± 3% RH in an environmental chamber before use in an assay. Adults were mixed ages but at least 24 h old.Field-captured Eucosma spp. through the primary literature [Eucosma spp.  was folded in half and inserted in a 271 cm long sticky card ring holder . The ring holder was bent at a 90° angle to wedge the card holder upright in position, which was subsequently wedged in the opening at the top of the PVC pipe. To protect against dislodgement by wind, sticky cards were affixed to the top of the metal card holder with a 50 mm × 30 mm binder clip. A single, capped LDPE 3-mL dropping bottle with one of the semiochemical treatments above was inserted in the top of the PCV pipe opening and affixed in place by tying it to the card holder with garden wire. Every week, the lures were replaced with a freshly prepared treatment and the position of the lure was rotated in the transect every two weeks. Traps were rotated because of the short duration of the flying season and resulted in every treatment occupying every position at least once. Sticky cards were changed on a weekly basis after the first recorded capture of an E. giganteana adult. Traps were deployed 7 June 2019 to 14 August 2019 and 15 June 2020 to 10 August 2020. In total, there were n = 3 replicates of each semiochemical treatment per field site. The number of E. giganteana and Lepidopteran nontargets was counted on each sticky card after freezing cards at −20 °C for at least 24 h.Each field had three transects spaced at least 10 m apart, each with a full set of semiochemical treatments represented. Each trap within the transect was spaced 10 m apart. Each trap consisted of a 1.27-cm diameter PVC pipe hammered in row with the silflower to a finished height of 1 m, in line with the canopy of n = 3 replicates per treatment and period combination.To confirm lure release rate over a two-week period, a volatile release rate experiment was performed, as has been conducted prior . Each ofAll headspace collection sample extracts were run on an Agilent 7890B gas chromatograph (GC) equipped with an Agilent Durabond HP-5 column  with He as the carrier gas at a constant 1.2 mL/min flow and 40 cm/s velocity, which was coupled with a single-quadrupole Agilent 5997B mass spectrometer (MS). The compounds were separated by auto-injecting 1 µL of each sample under split mode into the GC-MS at room temperature (approximately 25 °C). The flow was split in a 15:1 ratio with a split flow rate of 18 mL/min. The program consisted of 40 °C for 1 min followed by 10 °C/min increases to 300 °C and then held for 26.5 min. After a solvent delay of 3 min, mass ranges between 50 and 550 atomic mass units were scanned. Compounds were tentatively identified by comparison of their spectral data with those from the NIST 17 library and by GC retention index. Using the ratio of the peak area for the internal standard to the peak area for the other compounds in the headspace, the emission rates of samples in ng of volatile per μL of solvent and per h of collection were calculated. Identity of semiochemical lures and location on chromatogram was confirmed by injecting a diluted sample of a stock solution of pure compound.Six flight mills were situated on a 71.1 cm × 91.4 cm sheet of nonporous plastic to minimize vibrations. Each mill was spaced 39 cm apart in rows , and 25.4 cm between rows. Each mill contained three wires connected to the main connector board . A DC Power Supply  was set to the site and connected to the main wiring block to provide power. A 50-pin ribbon cable  ran from the main wiring connector block into a specially made PC port , where a computer with the software Labview 2017  automatically recorded the data from the mills. Each mill consisted of a PTFE cylinder with a head suspended by two antipodal magnets to create levitation and connected by an insect pin with lubricant to ensure frictionless turning. At the top of the head, 2 14 cm hollow tubes  extended perpendicularly with right angles formed at the end for insect attachment. Protruding from the bottom of the head was a magnet to activate the Halls Sensor placed on the base of the mill during rotation. Levels on the bottom of the mills and adjustable feet ensured that mills were even relative to center of gravity. Lighting was uniformly provided overhead using full spectrum lights.E. giganteana was descaled lightly with an artist’s paintbrush; then the copper loop was dipped in instant adhesive  and affixed onto the pronotum of the adult, ensuring not to impair proper wing functioning, and avoiding the eyes and antennae of the adults. The individual was tethered by inserting the point of the copper wire of the hypodermic needle attached to the rotation arm of each flight mill. Each trial was started between 15:00 and 18:00 by gently blowing on the insect to initiate flight and run for 24 h in parallel. Assays were conducted at 21.4 ± 0.01 °C temperature and 54.2 ± 0.2% RH and monitored with a datalogger . The semiochemical treatments in the flight mill assay included (E)-8-dodecenyl acetate, (Z)-9-dodecenyl acetate, and an unbaited control (acetone solvent only). Semiochemicals were freshly prepared in LDPE dropping bottles, as in the field-baiting experiment (as above), and placed in the center among the flight mills on a nonporous glass surface to prevent contamination. A smoke test confirmed that volatiles came into contact with each flight mill. Between runs, surfaces on the flight mill were thoroughly wiped down with solvent and allowed to dry and dissipate before use again. There were 12–18 replicates per treatment. At the end of a trial, insects were detached and weighed on a balance. Data were streamed in real time to a computer which automatically record the flight parameters: distance flown, the number of tandem flight bouts over the sampling interval (flights lasting more than 1 s), mean flight bout duration, and mean distance flown per bout. Data were also parsed by diurnal period  to determine maximum time of dispersal. Periods were defined as follows: Morning: 4:00–11:59; Afternoon: 12:00–19:59; Night: 20:00–3:59. Data were analyzed with R software.Six adults were run simultaneously on the six flight mills described above  to test flight capacity. A 14-gauge copper wire was stripped into individual threads and cut into 4 cm segments. The copper wire thread was wrapped around insect pin  embedded in modelling clay to form a loop. The loop was pinched with a standard metal forceps, then excess wire was ablated on the shorter end with scissors, and the loop was flattened relative to the plane of gravity. Moths were placed singly on a metal mason jar lid on top of ice and restrained with a plastic lid for a 59.1-mL container until sessile. Afterwards, the pronotum of E. giganteana or the number of Lepidopteran nontargets as the response variable. A first order autoregressive variance–covariance matrix was used to account for autocorrelation among sampling dates. Date was treated as a random variable. The fixed explanatory variable included semiochemical treatment  was used to analyze the data from the flight mill. In particular, the total distance flown in 24 h (km), number of flight bouts, duration of flight bouts (s), and distance flown in each flight bout (m) was coded as an aggregate response variable. Semiochemical treatment e.g.,  was usedE. giganteana moths were captured on sticky cards in 2019. The semiochemical lures significantly affected E. giganteana moth capture , with 2.5-fold more captures on sticky cards with (E)-8-dodecenyl acetate compared to the unbaited control (E)-8-dodecenyl acetate captured 36% of the total moths during the season (Z)-9-dodecenyl acetate was the second most attractive compound to E. giganteana, capturing 1.6-fold more moths when paired with clear sticky traps than the unbaited control (Z)-8-dodecenyl acetate, (E)-9-dodecenyl acetate, (Z)-8-dodecen-1-ol, and -8,10-dodecadien-1-yl acetate captured 28–35% of the moths found on the unbaited control sticky cards, suggesting repellency or active inhibition . Each semiochemical treatment captured 6–14% of the total nontargets on the sticky cards over the course of the season, with a mean number of 4–9 nontarget Lepidoptera per card . Like the prior year, there were 2.9-fold more captures of moths on sticky cards with (E)-8-dodecenyl acetate compared to the unbaited control (E)-8-dodecenyl acetate captured a third of the total moths during the season (Z)-8-dodecen-1-ol captured the fewest moths among the treatments, with zero captures the entire season, while -8,10-dodecadien-1-ol reduced captures on sticky cards by 88% compared to unbaited controls, suggesting active inhibition.In 2020, a total of 76  control . Moreovee season . Similar2 = 5.86; df = 8; p = 0.66). Each semiochemical treatment captured 10–14% of the total nontargets on the sticky cards over the course of the season, with a mean number of 3–5 nontarget Lepidoptera per card , ranging between 27 ± 9.7 and 45 ± 10 ng·h−1 (E)-9-dodecenyl acetate and -8,10-dodecadien-1-ol were numerically the highest and lowest emitter, respectively. Emissions lasted over the full sampling period.The mean emission rates among the lures used in the field baiting experiment were statistically similar over a 14-d period  and the interaction between sex and semiochemical . As a result, sequential ANOVAs were performed for each response variable.Overall, the semiochemical present significantly affected the flight behavior of E. giganteana over 24 h . For example, the distance flown by E. giganteana was only approximately half as much in the presence of (Z)-9-dodecenyl acetate compared with no stimuli , with females flying 1.5-fold farther on average . By contrast, neither the semiochemical treatment , sex , nor their interaction  affected the total number of flight bouts by E. giganteana . For instance, in the presence of (Z)-9-dodecenyl acetate and (E)-8-dodecenyl acetate, moths only flew 18% and 44% of the duration that control moths flew when no stimuli were present , nor did its interaction with semiochemical .The semiochemical present significantly affected the mean duration of each flight bout by  present . HoweverF = 30.6; df = 2, 36; p < 0.0001), with only 19–22% of the distance flown when (Z)-9-dodecenyl acetate and (E)-8-dodecenyl acetate were present compared to the unbaited control , with female moths flying 2.1-fold farther than males on average. The interaction between sex and semiochemical treatment significantly affected the distance flown in each bout . For example, distance flown in each bout by males was marginally reduced in the presence of (E)-8-dodecenyl acetate, while, for females, it was more reduced by (Z)-9-dodecenyl acetate compared to control moths.Furthermore, the semiochemical present significantly affected the distance flown in each flight bout , and its interaction with the period  significantly affected the flight behavior of E. giganteana. As a result, sequential ANOVAs were performed for each response variable.Overall, the period significantly affected the flight behavior of F = 1.44; df = 2, 99; p = 0.24) by E. giganteana, the semiochemical lure did . By contrast, neither the period , semiochemical lure , nor their interaction  affected the total number of flight bouts by E. giganteana , with E. giganteana moths flying 1.8-fold longer in the morning than in the night , but its interaction with period was not significant .The flight duration of each bout was significantly affected by period , with moths flying 1.8-fold farther in the morning than in the night , but its interaction with period was not significant .Similarly, the total distance flown by he night . In addiE. giganteana. In both years of the field study, (E)-8-dodecenyl acetate, and to a lesser extent, (Z)-9-dodecenyl acetate, acted as an attractant when paired with a clear sticky card in the field for E. giganteana. The most attractive stimulus reliably captured moths throughout their period of flight each year. Both compounds and other structurally related chemicals have shown biological activity for related Eucosma species in prior studies. For example, Rhyacionia zozana (Kearfott) (Lepidoptera: Olethreutidae) was attracted to the closely related (E)-9-dodecenyl acetate, whereas Eucosma sonomana was attracted to a mix of the Z- and E- isomers of this compound [E)-9-dodecenyl acetate when used in a mating disruption strategy successfully suppressed damage by E. sonomana in 19 ha of forest, reducing damage by 67 to 79% [Eucosma coniogramma was described as (E)-9-dodecenyl acetate as the major component, which was attractive to conspecifics in the field and produced a robust GC-EAD response [E)-8-dodecenyl acetate to (Z)-8-dodecenyl acetate on traps significantly decreased trap captures of Eucosma notanthes [Z)-8-dodecenyl acetate and (Z)-8-dodecenol have been described as the two-component sex pheromone for E. notanthes [Eucosma monitorana Heinrich, Eucosma gloriola Heinrich, and Eucosma tocullionana Heinrich were attracted to lures containing (Z)-9- and (E)-9-dodecenyl acetate in various ratios with or without (Z)-9-dodecen-1-ol [Eucosma womonana Kearfott was captured by traps baited with a blend of five acetates, including (Z)-5-dodecenyl acetate and (Z)-7-dodecenyl acetate [Grapholita molesta (Busck) (Lepidoptera: Tortricidae), has been described as a combination of (Z/E)-8-dodecenyl acetates [Z)-8-dodecen-1-ol, has proven highly effective when implemented using SPLAT technology for mating disruption of the species in apple orchards [Eucosma spp., which is why 5 were included among the semiochemical treatments in this study. Our study strongly suggests that (E)-8-dodecenyl acetate is a component of E. giganteana pheromone blend, although it is likely a minor one given a small 3-fold increase in attraction over control. Furthermore, it attracted the highest number of E. giganteana moths in both years and consistently over the course of the growing season. Future work should determine the other components of the pheromone blend and evaluate whether it can be used for mating disruption.This is the first study to investigate behaviorally relevant semiochemicals and the flight behavior of compound . In fact7 to 79% . The sexresponse . Howeverotanthes . Rather,otanthes . In pinecen-1-ol . In the  acetate . Notablyacetates , and a borchards . OverallE)-8-dodecenyl acetate, the peak periods of flight of E. giganteana were 28 June to 9 August in 2019 and 15 June to 3 August 2020. While other work has monitored larval development through the season for E. giganteana [E. gloriola, E. monitorana, and E. tocullionana were from 8 May to 4 June, 15 May to 26 June, and 22 May to 26 June 1991 in Ontario, Canada [Eucosma scorzonerana was shown to have a flight period from the end of May to the middle of June in Sweden [E. giganteana and lasts longer than for other Eucosma species at higher latitudes.According to trap catches with . To assess whether E. giganteana is a random walker, release–recapture studies should be performed to see the maximum distance that they can fly from release points around a trap. Males appeared to be preferentially arrested by the presence of (E)-8-dodecenyl acetate, compared to females, which were more arrested in flight behavior by (Z)-9-dodecenyl acetate. A potential explanation for this may be because the former compound more closely resembles the natural sex pheromone, while the latter may indicate competing species and availability of preferred oviposition sites. Thus, for separate proximal reasons, both males and females exhibit shorter flights and arrestment when exposed to these compounds. There have been no prior studies on the flight behavior of E. giganteana, although E. monitorana was described as a weak flyer [E. giganteana appears to be a strong flyer, capable of extended, directed flight -8-dodecenyl acetate and (Z)-9-dodecenyl acetate had an arresting function on the behavior of E. giganteana. Both compounds appear to affect the flight behavior of E. giganteana. Interestingly, (Z)-9-dodecenyl acetate was just as arresting, even though it was not nearly as attractive in the field baiting experiment. This raises the possibility that blends of the semiochemicals used in this study may have synergistic properties. In a meta-analysis, Szendrei and Rodriguez-Saona [E. giganteana to determine if there is a synergistic response.The presence of attractive stimuli reduced the distance flown in each flight bout by an average of 78 to 80%. This suggests that both (ez-Saona  found thE. giganteana in both duration and distance occurred in the morning (4:00–12:00). Adult E. giganteana flew 1.8-fold farther in each flight bout and 1.8-fold longer in the morning than at night. In other related species, adult Eucosma obumbratana are primarily active at sunset [E. scorzonerana are not only active at sunset but also in early morning and late afternoon [E. giganteana, it may be reasonable to expect that mate-finding would drive flight behavior during the period of peak activity.Although the total distance flown was not affected by the flight period, the longest flight bouts of t sunset ,36, wherfternoon . Night-tE. giganteana occurs at high densities, with multiple individuals per plant often widely spaced, making possible pheromone autodetection in this species. However, patches of silflower may be limiting, and thus the autodetection of pheromone by females may result in arrestment instead of flight because it could be an indicator of a feeding patch with other mating and oviposition opportunities that are relatively rare in the environment. As a result, both males and females were included in the study.We have included tests of both male and female response to potential pheromones in this study. Historically, female moths and their response to pheromones have been understudied component of the literature compared to their male counterparts. Prior work has shown that female autodetection of pheromone occurs in two important agricultural tortricids, affecting their flight behavior and having important implications in mating disruption programs . One potZ)-8-dodecen-1-ol was also behaviorally active and consistently acted as a repellent to captures of E. giganteana on sticky cards. This information may be helpful, once the pheromone is identified, to know what trace impurities may hinder attraction to synthetic lures. In addition, depending on the behavioral dynamics displayed by E. giganteana in the presence of this compound, it may serve as a suitable semiochemical for incorporation in a push–pull strategy [Among the other volatiles in this study,  collect headspace from E. giganteana conspecifics and perform glandular microdissections to identify the true sex pheromone, (2) confirm antennal response by E. giganteana to the compounds cited in this study by GC-EAD, and (3) evaluate combinations of 12-carbon acetates for synergistic response by moths. Furthermore, future work should relate trap captures of E. giganteana to S. integrifolium crop damage to develop pest management action threshold guidelines. This would be an additional tool in the IPM toolkit against E. giganteana. While this work has focused on testing congeneric pheromonal stimuli for behavioral response by E. giganteana, it is likely that host plant volatiles from S. integrifolium are also important for attraction by conspecifics. However, little is known about the headspace volatile profiles of silflower, or how it may influence the behavior of E. giganteana, so future work ought to include a rigorous evaluation of host plant volatiles. Ultimately, we have demonstrated that multiple volatiles may be useful in manipulating the flight behavior of E. giganteana and, when paired with clear sticky traps, provide a new IPM tool for monitoring E. giganteana populations.To our knowledge, this study provides new information about the chemical ecology, flight behavior, and dispersal capacity of


Efforts to resection of glioma lesions located in brain-eloquent areas must balance the extent of resection (EOR) and functional preservation. Currently, intraoperative direct electrical stimulation (DES) is the gold standard for achieving the maximum EOR while preserving as much functionality as possible. However, intraoperative DES inevitably involves risks of infection and epilepsy. The aim of this study was to verify the reliability of individual-target transcranial magnetic stimulation (IT-TMS) in preoperative mapping relative to DES and evaluate its effectiveness based on postsurgical outcomes.nTMS). Both groups then underwent awake surgery and intraoperative DES mapping. DES hotspots (HSDES) were also determined in a manner analogous to HSnTMS. The spatial distribution of HSnTMS and HSDES in the nTMS group was recorded, registered in a single brain template, and compared. The center of gravity (CoG) of HSnTMS (HSnTMS-CoG)-based and HSDES-CoG-based diffusion tensor imaging-fiber tracking (DTI-FT) was performed. The electromagnetic simulation was conducted, and the values were then compared between the nTMS and DES groups, as were the Western Aphasia Battery (WAB) scale and fiber-tracking values.Sixteen language-eloquent glioma patients were enrolled. Nine of them underwent preoperative nTMS mapping , and the other seven were assigned to the non-nTMS group and did not undergo preoperative nTMS mapping (n=7). Before surgery, online IT-TMS was performed during a language task in the nTMS group. Sites in the cortex at which this task was disturbed in three consecutive trials were recorded and regarded as positive and designated nTMS hotspots . A higher fractional anisotropy (FA) value in nTMS mapping (P=0.0373) and an analogous fiber tract length (P=0.2290) were observed. A similar distribution of the electric field within the brain tissues induced by nTMS and DES was noted. Compared with the non-nTMS group, the integration of nTMS led to a significant improvement in language performance  as well as in brain-structure preservation .HSPreoperative IT-TMS provides data equally crucial to DES and thus facilitates precise brain mapping and the preservation of linguistic function. GeneralIn order to map the language cortex preoperatively, a new technique combining advanced imaging and electrophysiology, namely navigated transcranial magnetic stimulation (nTMS), has been introduced , 14. TraIndividual target (IT)-TMS, a novel form of nTMS that combines the personalized stimulation site and dose with precise robotic targeting, has already been shown to increase the remission rates of major depressive disorders significantly compared with intermittent theta burst stimulation  as well 22.1This study included sixteen patients, 6 men and 10 women aged 28–69 , with brain glioma located in areas surrounding classic Broca’s area, inferior frontal gyrus and ventral precentral gyrus. All surgeries were performed by the same surgeon, who commonly finishes about 150 glioma surgeries every year in his department. All of the subjects provided written informed consent. All of the procedures in this study were approved by the ethics committee of Xijing Hospital and conducted under the guidelines of the Declaration of Helsinki.2.2The subjects received preoperative and postoperative awake MRI scans, including T1, T2, and DTI. The scans were performed using a 3.0 T scanner equipped with a 32-channel head coil. T1-weighted sagittal anatomical images were obtained with the following parameters: sagittal slices = 192; repetition time (TR) = 7.24 ms; echo time (TE) = 3.10 ms; slice thickness/gap = 0.5/0 mm; in-plane resolution = 512 × 512; inversion time (TI) = 750 ms; flip angle = 10°; field of view (FOV) = 256 × 256 mm; voxel size = 0.5 × 0.5 × 1 mm; and T2 with TR/TE/FOV/voxel size/slice number 2,500 ms/236 ms/240 mm/1 mm × 1 mm × 1 mm/200. The DTI data were acquired with the repetition time (TR) = 12,676 ms; echo time (TE) = 88.6 ms, slice thickness = 2 mm, flip angle = 90°. All subjects wore earplugs to reduce the noise and possible head motion.2.3nTMS). The resting motor threshold  was measured over the abductor pollicis brevis (APB).We used the Black Dolphin Navigation Robot  with a figure-of-8 coil  to perform the IT-TMS. An infrared camera and a three-dimensional individual mask were used for precise navigation of the coil over the target area under real-time visualization. The possible individual language sites close to the lesions of each subject in the nTMS group were marked prior to the experiments. Briefly, based on individual MRI brain images, the predefined targets were integrated into the operation system, in which a 3D restoration of the brain images and targets was visualized that allowed for the visual selection and monitoring of the immediate targets selection and monitoring. Next, online continuous theta burst stimulation (cTBS) examining these sites was performed before the subjects entered the operation room, with a number-counting task being performed in the meantime. When language was disturbed, the site was regarded as an nTMS hotspot (HS2.4DES).For each subject in the two groups, an awake craniotomy was performed to gain access to the tumor regions near the language areas, during which procedure the language task and DES targeting the language cortex were conducted to test whether language was disturbed. The DES was guided by the results of the preoperative data provided by the nTMS and nTMS-based DTI-FT (when available). A single anodal square pulse (pulse duration 0.2 ms) was employed. The minimum intensities from 1 mA, 2 mA to maximally 6 mA were applied when no response was achieved. When language was disturbed in three consecutive trials , 30, the2.5Given the uniformity of enrolled subjects, we aimed to identify language-eloquent sites for speech production. Hence, only number-counting task was performed during the nTMS and DES procedures , 31, 32.2.6σskin=0.465 S/m, σskulll=0.010 S/m, σCSF=1.654 S/m, σGM=0.2765 S/m, σWM=0.126 S/m The electrical conductivity of glioma tissue was consistent with that of surrounding gray matter.SimNIBS software (Version 3.2.4) served to generate for each subject the finite element mesh model based on the T1 images. A 3Dslicer served to segment the tumor using the intensity threshold method, and manual correction was performed. The isotropic tissue conductivity was as follows: For the nTMS, SimNIBS software again served to simulate the electric field. The coil model was established by measuring the magnetic field from the center of gravity (CoG). For the DES, the monopolar electrical stimulation was modeled by applying a Dirichlet boundary condition  for the 2.7nTMS) included in the area of the DES-induced electric field (EDES) in a DES-determined region of interest (ROI). In the next step we computed the CoG of HSnTMS (HSnTMS-CoG) and HSDES-CoG for each subject as described previously  values were predefined as 0.1 as well as 50% of the individual FA threshold, as is conventionally done for the purpose of fiber tracking  scale  as the p2.10The parameters assessed in this study are presented as means ± standard deviations and calculated using SPSS software. GraphPad Prism software served to generate the values and graphs. We performed all of the computations described here for each subject individually and then calculated the mean over all of the subjects.33.1First, preoperative IT-TMS mapping was performed on one subject, as shown in 3.2nTMS-CoG), and the blue dot indicates the HSDES-CoG.The distribution of positive nTMS language mapping sites was similar to those of DES Figure 23.3P=0.2290, P=0.0373, As 3.4nTMS-CoG with the HSDES-CoG area for each subject in the nTMS group . Postoperatively, the mean WAB score in nTMS group was , and in non-nTMS group . The independent-samples t-test between these two groups showed a P value of 0.0321 (less than 0.05). Moreover, according to the DTI-FT of AF and SLF-II + III results shown in As shown in 4The management of gliomas close to functional areas is challenging because of the risk of surgery-related morbidity . Thus, fOther researchers have explored the effective use of nTMS mapping in surgical techniques with respect to such considerations as the scope of craniotomy , gross tThe fiber tracts, AF and SLF-II + III overlap in the classic Broca’s area and ventral precentral gyrus, are the main structures responsible for language production , 44. DESThe analysis of the distribution of the electric fields that nTMS and DES induce yielded another key finding from this study. In fact, the cortical and subcortical currents, namely virtual lesions , originaRegarding effectiveness, the findings of a higher WAB score and better DTI-FT value Table 2 This study thus provides evidence that preoperative nTMS correlates well with intraoperative DES in language-eloquent mapping and, therefore, contributes to good language performance for language-eloquent glioma patients after surgery. The electromagnetic simulation results reveal a comparison between nTMS and DES, demonstrating that the brain tissues on or beneath the cortex receive the equivalent level of electric energy. Above all, preoperative nTMS mapping is a specific non-invasive method which does not require a lengthy operation and might significantly reduce the surgical risk of infections and limitations of other complications.5This study has several limitations. First, the small sample size and restriction of the population to glioma patients may have introduced bias. For precise brain-mapping validation, future research could be conducted with larger samples that include healthy subjects and/or subjects with other brain diseases. Second, we did not determine whether IT-TMS-based language mapping alone improves clinical outcomes. Lastly, we did not compare mapping based on IT-TMS only and DES only in this study because of a problem with the ethical review.In conclusion, we have described here the reliability and effectiveness of preoperative IT-TMS-based brain functional language mapping. In doing so, we provide novel evidence of fiber-tracking and electromagnetic simulation for the preoperative neurophysiological mapping of language sites prior to surgery to treat intrinsic brain tumors located in or near eloquent networks.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethnic review board of Xijing Hospital. The patients/participants provided their written informed consent to participate in this study.SL and YR designed this study and directed the trial. CS collected and analyzed the MRI data. XL and HL prepared the algorithm. XuY recruited the subjects and collected the clinical information. XiY performed the electromagnetic simulation. YD conducted the DTI-FT and statistical analysis. YW and YR prepared the manuscript. ZF and SL are the co-corresponding authors; they directed the research program, revised the draft of the manuscript, and submitted this article. All authors contributed to the article and approved the submitted version.


Thermotoga spp. are promising candidates for the metabolic engineering of cellulosic ethanol producing strains. However, Thermotoga spp. are hydrogen producers, and none of them have been found to produce significant amount of ethanol. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from ac-CoA to ethanol. Aldehyde dehydrogenases (Aldhs) and alcohol dehydrogenases (Adhs) are the vital enzymes involved in the metabolism of ethanol in Thermotoga spp.. However, the biochemical properties of a number of Adhs related to ethanol formation and consumption have not been identified until now. The aim of this study was to determine the role of putative Adhs in T. neapolitana (Tne) in the pathway from ac-CoA to ethanol. We found that the Fe-AAdh (encoded by CTN_0580) and Fe-Adh2 (encoded by CTN_1756) were the main enzymes responsible for ethanol production in Tne. While Zn-Adh (encoded by CTN_0257) was identified as the main protein responsible for ethanol consumption in the acid environment and Fe-Adh1 (encoded by CTN_1655) played a minor role in ethonal production and consumption in Tne. Basing on these results, it will be helpful to construct and assemble a novel hyperthermophilic strain to produce cellulosic ethanol.Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.Hyperthermophilic  Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis are considered as the main ethanol-producing strains because a high ethanol concentration and yield can be achieved via metabolic engineering [Z. mobilis is one of the most typical strains for two-step ethanol fermentation for its high ethanol productivity, ethanol tolerance, and genetic manipulation feasibility [E. coli to improve ethanol production [S. cerevisiae without engineering [S. cerevisiae and Z. mobilis cannot usually metabolize pentose sugars unless they are engineered properly. All of these limitations, including the requirement of a complex pretreatment and co-expression of heterologous genes in the target strains, will impede bioethanol production.Biomass (lignocellulose) is widespread in nature, and has been in the limelight for second-generation bioethanol production, a promising alternative to fossil fuels. Therefore, it is not surprising that the metabolism and bioconversion of biomass attracts much attention ,2. Escheineering ,4,5. Z. ineering . Howeverineering ,8. On thThermoanaerobacterium, Thermoanaerobacter, and Clostridium, which have good ethanol yields [C. thermocellum was enhanced significantly by introducing adhA, adhE, and nfnAB simultaneously from T. saccharolyticum [Thermoanaerobacterium aotearoense SCUT27/Δldh/ΔpflA, which was grown anaerobically at an initial pH of 6.5 and 55 °C [Thermophilic anaerobic bacteria have received increasing interest as a promising bioprocessing platform for bioethanol production due to their broad substrate range and higher operating temperature, like bacteria within the genera of l yields ,10,11,12olyticum . The finnd 55 °C .Thermotoga spp. could secrete a series of thermostable cellulases and hemi-cellulases, and could also grow at 80 °C (near the ethanol distillation temperature), which make Thermotoga spp. more suitable for integrating these units into consolidated bioprocessing in one pot and potentially reducing the operating cost. Cooling is considered to be a significant cost of the industrial fermentation process (mesophilic), so this may not be needed for the process at high temperatures, at which, the separation of bioethanol may be carried out in situ [Thermotoga spp. are also cellulolytic and hemicellulolytic hyperthermophiles that meet the requirements for the construction of a cellulosic biofuel producer. Hydrogen production by Thermotoga spp. was tested using soluble starch or CMC or cellulose as the carbon source. The strains showed a 39–49 mL H2/L culture from cellulose, 172–175 mL H2/L culture from CMC, and 345–404 mL H2/L culture from soluble starch [Hyperthermophilic anaerobic bacteria  are considered to be one of the most promising candidates for the production of bioethanol from complex lignocellulosic biomass, with great advantages of a broad substrate range, higher operating temperatures, higher ethanol diffusion rates, and lower cooling and distillation costs, as well as a lower risk of microbial contamination ,15. Unit in situ ,18,19. Te starch .Pyrococcus furiosus and the engineered stain produced more than 20 mM ethanol via acetate and acetaldehyde [T. maritima and T. hypogea were found to have CoA-dependent PDC activity, where the PDC activity is less than 2% of POR activity [Researchers have been focusing their efforts on aldehyde dehydrogenase (Aldh) and Adh since 1988 ,22. The aldehyde . The pyractivity ,25,26. Tactivity .Thermotoga spp. were characterized and the crystal structure was obtained [Thermotoga spp. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from ac-CoA to ethanol. Aldehyde dehydrogenases (Aldhs) and alcohol dehydrogenases (Adhs) are the vital enzymes involved in the metabolism of ethanol in Thermotoga spp. However, the biochemical properties of a number of Adhs related to ethanol formation and consumption have not been identified until now. The aim of this study was to determine the role of putative Adhs in T. neapolitana in the pathway from ac-CoA to ethanol.It is true that some alcohol dehydrogenases from obtained . HoweverE. coli JM109 (Promega) was used as host for cloning and expression of genes in a pHsh vector constructed by this laboratory . E. coli cells were routinely grown aerobically in Luria–Bertani (LB) medium at 30 °C, and 100 μg/mL ampicillin was added to the LB medium for selective cultures. The plasmids pHsh-1655, pHsh-1756, and pHsh-0257 were constructed previously [eviously .E. coli JM109 cells were transformed using the expression plasmids containing the Adh genes and were grown at 30 °C to an OD600 of 0.6–0.8 before the cultures were transferred to a 42 °C shaking water-bath incubator for heat-shock induction of gene expression. After 6 h of cultivation along with gene expression at 42 °C, cells were harvested and transferred into an anaerobic chamber for the isolation and purification of recombinant enzymes. E. coli cells were resuspended in degassed 3-(N-morpholino) propanesulfonic acid buffer  and disrupted by sonication, followed by heat treatment at 75 °C for 30 min.g, 30 min), the supernatants were mixed with the same volume of 2× binding buffer and loaded to the Ni-metal affinity column (Novagen) according to the manufacturer’s instructions. Each recombinant enzyme bound to Ni-column was eluted and collected into a dialysis bag, concentrated by embedding the dialysis bag in PEG 20,000, and dialyzed against MOPS with two changes. Purified enzymes were stored in MOPS with the addition of 10% glycerol, 1 mM dithiothreitol (DTT), 0.02% (w/v) NaN3, and 1 mM FeCl2 or ZnCl2 for Fe-Adhs or Zn-Adh, respectively.After the cell debris and heat-denatured protein were removed by centrifugation .Protein expression levels and the purity of recombinant proteins were examined by SDS-PAGE . Protein+ or NAD(P)H, and 20 mM acetaldehyde or ethanol, at different pH (4–10) and temperatures (50–100 °C) as indicated for each reaction.Adh activities were measured by monitoring the changes in NAD(P)H absorbance at 340 nm. One unit of enzyme activity was defined as the amount of enzyme producing or consuming 1 μmol of NAD(P)H per min. All of the activity assays were performed anaerobically in triplicate, and data reported here are the mean of the three replicates. For standard enzyme activity assays, the reaction mixture consisted of 100 μL of MOPS containing suitable substrates at concentrations: 1 mM NAD(P)T. neapolitana genes CTN_1655, CTN_1756, and CTN_0257 were designated as Fe-Adh1, Fe-Adh2, and Zn-Adh, respectively. These enzymes were purified to near gel electrophoresis homogeneity after heat treatment and metal affinity chromatography using a Ni-column at room temperature. However, the purified Fe-Adh1 and Fe-Adh2 became inactivated within approximately one week after they were stored at −20 °C, implying that they were sensitive to O2 in the air and buffer. This problem was resolved by purifying these enzymes in an anaerobic chamber using degassed buffers and keeping the purified enzyme in sealed anaerobic serum tubes.The plasmids pHsh-1655, pHsh-1756, and pHsh-0257 were constructed previously . The recThe biochemical properties of enzymes were first characterized at different reaction pH and temperatures. Fe-Adh1, Fe-Adh2, and Zn-Adh did not show any ac-CoA reduction activity . However+, NADP+ was preferably served as the cofactor by the three Adhs. Although the specific activity of Zn-Adh was similar to that of Fe-Adh1 and much lower than that of Fe-Adh2, the reverse reaction would probably be driven by Zn-Adh due to the near physiological pH.The cofactor dependence of these enzymes was characterized at their optimal pH and temperatures in each step . When anT. neapolitana Zn-Adh specifically catalyzed the reaction from ethanol to acetaldehyde, and no activity was detectable for the reaction from acetaldehyde to ethanol. This result is different from almost all of the other Adhs reported previously [T. neapolitana Zn-Adh reacted optimally at pH 6.0, which was close to pH 6.5, the pH value of non-buffered cell-free extracts of T. neapolitana. The CoA-independent Aldh (encoded by CTN_1548) had an optimal pH of 9.5 (data not shown), which, together with Zn-Adh, could possibly form a detoxification system for the oxidation of ethanol and aldehyde.The last two steps of ethanol fermentation in anaerobes are two reversible redox reactions, from ac-CoA to acetaldehyde catalyzed by the CoA-dependent aldehyde dehydrogenase (Aldh), and from acetaldehyde to ethanol catalyzed by Adh. Fe-AAdh was the first enzyme found in hyperthermophiles that catalyzed the reduction of ac-CoA to produce acetaldehyde . Fe-Adh1eviously ,22,30. TT. maritima and T. hypogea were found to convert 98% of pyruvate to ac-CoA [Ethanol is an important renewable fuel that could be produced from pyruvate via PDC, or from ac-CoA via bifunctional aldehyde/alcohol dehydrogenase (such as AdhE) . At preso ac-CoA ,26. The o ac-CoA ,33,34,35T. neapolitana were characterized at their optimal reaction pH and temperatures . However, there is strong Fe-hydrogenase to utilize ferredoxin and NADH synergistically for anaerobic H2 production so that H2 becomes the main fermentation product of Thermotoga spp. [Hyperthermophilic oga spp. .Thermotoga spp. include acetate, lactate, and ethanol. Metabolic engineering has proven to be an effective approach to constructing novel ethanol-producing strains. By the heterologous expression of a thermostable acetyl-CoA synthetase that catalyzed the irreversible acetate assimilation in T. neapolitana, a strongly increased lactate production (up to 2.5 g/L) was achieved by the recombinant stain from acetate and CO2 [adhA and adhE from Thermoanaerobacter, the highest amount of ethanol  was produced in P. furiosus [Caldanaerobacter subterraneus resulted in a high ratio of ethanol over acetate (>8:1) and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date [The major fermentation non-gaseous end-products of  and CO2 . These e and CO2 ,39 or hy and CO2 . The sec and CO2 ,42. For furiosus . The ins to date .2 and lactate formation, improving the activity of bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh), or even strengthening the coenzyme (NADPH) cycles, it will be a novel strategy for the construction and assembling of a hyperthermophilic strain to produce cellulosic ethanol.The cellulosic ethanol fermentation at temperatures above the ethanol boiling point has advantages in reducing the costs for autoclaving raw-materials and distilling ethanol from fermentation, which also favors the consolidate process of biomass degradation, ethanol fermentation, and distillation ,15. By eT. neapolitana genome were investigated for their roles in ethanol metabolism for the first time. We report that the Fe-AAdh (encoded by CTN_0580) and Fe-Adh2 (encoded by CTN_1756) were the main enzymes responsible for ethanol production in T. neapolitana while Zn-Adh (encoded by CTN_0257) was identified as the main protein responsible for ethanol consumption in the acid environment and Fe-Adh1 (encoded by CTN_1655) played a minor role in ethanol production and consumption in T. neapolitana.In this study, three putative Adh genes in the